Pipecolinic acid 4-hydroxylase and method for producing 4-hydroxy amino acid using same

ABSTRACT

The present invention provides a pipecolic acid 4-hydroxylase protein exemplified by the following (A), (B), and (C), having activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions to produce trans-4-hydroxy-L-pipecolic acid, and a method for producing 4-hydroxy amino acid, which method comprises reacting the pipecolic acid 4-hydroxylase protein, cells containing the protein, a treated product of the cells, and/or a culture liquid obtained by culturing the cells, with α-amino acid to produce 4-hydroxy amino acid:
     (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18;   (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and   (C) a polypeptide having an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.

TECHNICAL FIELD

The present invention relates to a method for producing 4-hydroxy amino acid, which method utilizes a novel pipecolic acid 4-hydroxylase.

BACKGROUND ART

Amino acid hydroxylases are enzymes useful for production of intermediates of pharmaceuticals and the like, and the presence of proline 4-hydroxylase (Non-patent Document 1), L-isoleucine dioxygenase (Non-patent Document 2), and the like has been reported. In terms of enzymes having an ability to hydroxylate pipecolic acid, several kinds of proline hydroxylases have been reported to have an ability to hydroxylate 3-position or 5-position of L-pipecolic acid (Non-patent Document 3). However, no enzyme has so far been reported to hydroxylate 4-position of pipecolic acid.

The amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, and 12 are the same as GenBank accession NOs. EGU81245, XP_001827566, XP_002558179, XP_383389, ELA34460, and XP_659994, respectively, which are the amino acid sequences translated from the putative protein-coding DNA sequences based on genomic sequence information of the Fusarium oxysporum Fo5176 strain, Aspergillus oryzae RIB40 strain, Penicillium chrysogenum Wisconsin 54-1255 strain, Gibberella zeae (another name, Fusarium graminearum) PH-1 strain, Colletotrichum gloeosporioides Nara gc5 strain, and Aspergillus nidulans (another name, Emericella nidulans) FGSC A4, respectively. Since all of these proteins are derived from fungi, the proteins, if exist, are likely to be in a state where they have undergone glycosylation after their expression. However, there has been no report supporting their existence based on actual isolation or the like, and the functions of these proteins have been unknown at all.

Optically active 4-hydroxy amino acids are useful substances as intermediates of pharmaceuticals and the like. For example, (4S)-hydroxy-L-pipecolic acid can be used as a precursor of a Rho kinase inhibitor (Patent Document 1), and (4S)-hydroxy-D-pipecolic acid can be used as a precursor of CGS-20281, which is an NMDA receptor inhibitor (Non-patent Document 4). (4R)-Hydroxy-L-proline can be used as a precursor of icatibant acetate, which is a bradykinin B2 receptor inhibitor (Non-patent Document 5), and (4R)-hydroxy-D-proline can be used as a precursor of a factor Xa inhibitor (Patent Document 2). L-Homoserine can be used as a precursor of Omapatrilat, which is an ACE inhibitor (Patent Document 3), and 4-hydroxy-L-leucine can be used as a precursor of SCY-635, which is a cyclophilin inhibitor (Non-patent Document 6).

Examples of previously reported methods for synthesis of an optically active 4-hydroxy amino acid include a method in which (4S)-hydroxy-L-pipecolic acid or (4S)-hydroxy-D-pipecolic acid is produced by stereoselective cyclization of 3-butenol with an optically active C-aminocarbonyl nitrone or C-alkoxycarbonyl nitrone has been reported so far (Non-patent Document 7). A method in which (4R)-hydroxy-L-proline is synthesized from L-proline using proline 4-hydroxylase derived from the Dactylosporagium RH 1 strain (Non-patent Document 1), and a method in which (4R)-hydroxy-D-proline is synthesized via an α,β-didehydroamino acid (Non-patent Document 8) have also been reported. A method in which L-homoserine is synthesized by fermentation using a recombinant E. coli (Patent Document 4), and a method in which 4-hydroxy-L-leucine is synthesized from L-leucine using L-isoleucine dioxygenase derived from the Bacillus thuringiensis 2e2 strain (Non-patent Document 2) have also been reported.

However, more efficient synthesis methods have been demanded since these methods have problems such as expensive materials, large numbers of steps, small numbers of the types of compounds to which the methods are applicable, and high purification loads.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Translated PCT Patent Application     Laid-open No. 2010-514720 -   Patent Document 2: Japanese Translated PCT Patent Application     Laid-open No. 2009-526813 -   Patent Document 3: JP 7-48259 A -   Patent Document 4: WO 2013/134625

Non-Patent Documents

-   Non-patent Document 1: Shibasaki et al., Appl. Environ. Microbiol.,     1999, 65, 4028 -   Non-patent Document 2: Hibi et al., Appl. Environ. Microbiol., 2011,     77, 6926 -   Non-patent Document 3: Klein et al., Adv. Synth. Catal., 2011, 353,     1375 -   Non-patent Document 4: Occhiato et al., SYNTHESIS, 2009, 3611 -   Non-patent Document 5: Bork et al., Nat. Rev. Drug Discov., 2008, 7,     801 -   Non-patent Document 6: Hopkins et al., Antimicrobial. Agents     Chemother., 2010, 54, 660 -   Non-patent Document 7: Cordero et al., Eur. J. Org. Chem 2006, 3235 -   Non-patent Document 8: Kimura et al., Bull. Chem. Soc. Jpn., 2002,     75, 2517

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel pipecolic acid 4-hydroxylase, and a novel method for inexpensively and simply producing a 4-hydroxy amino acid, especially an optically active 4-hydroxy amino acid.

Means for Solving the Problems

In order to solve the problems described above, the present inventors intensively studied on a method for producing an optically active 4-hydroxy amino acid. As a result, the present inventors discovered that the protein FOXB_08233 derived from the Fusarium oxysporum Fo5176 strain, whose isolation as a protein has not been reported and whose function has been unknown, and its homologue proteins, have a 2-oxoglutaric acid-dependent pipecolic acid 4-hydroxylase activity. The present inventors then discovered that, by preparing transformants using DNAs encoding these proteins and reacting the prepared transformant cells, treated products thereof, and/or culture liquids thereof with various amino acids, various optically active 4-hydroxy amino acid can be obtained at high optical purity. The present invention was achieved based on these discoveries.

That is, the present invention can be summarized as follows.

[1] A pipecolic acid 4-hydroxylase protein having activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions, to produce trans-4-hydroxy-L-pipecolic acid. [2] The pipecolic acid 4-hydroxylase protein according to [1], selected from the group consisting of the following (A), (B), and (C):

(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18;

(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and

(C) a polypeptide comprising an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.

[3] The pipecolic acid 4-hydroxylase protein according to [2], which is produced as a recombinant protein by a host having no glycosylation ability. [4] A method for producing 4-hydroxy amino acid, the method comprising reacting the pipecolic acid 4-hydroxylase protein according to any one of [1] to [3], a cell(s) comprising the protein, a treated product of the cell(s), and/or a culture liquid obtained by culturing the cell(s), with α-amino acid to produce 4-hydroxy amino acid. [5] The method for producing 4-hydroxy amino acid according to [4], wherein the α-amino acid is represented by the following General Formula (I):

(wherein each of R¹, R², R³, R⁴, and R⁵ represents a hydrogen atom or C₁-C₃ alkyl, and alternatively R² may bind to R⁵ or the nitrogen atom of the amino group to form a ring structure), and the 4-hydroxy amino acid is represented by the following General Formula (II):

(wherein each of R¹, R², R³, R⁴, and R⁵ represents a hydrogen atom or C₁-C₃ alkyl, and alternatively R² may bind to R³ or the nitrogen atom of the amino group to form a ring structure). [6] The method for producing 4-hydroxy amino acid according to [4], wherein the α-amino acid is pipecolic acid, and 4-hydroxy amino acid is 4-hydroxy-L-pipecolic acid. [7] The method for producing 4-hydroxy amino acid according to any one of [4] to [6], wherein the cell comprising pipecolic acid 4-hydroxylase is a cell transformed with a DNA encoding pipecolic acid 4-hydroxylase protein. [8] The method for producing 4-hydroxy amino acid according to [7], wherein the DNA encoding pipecolic acid 4-hydroxylase protein is selected from the group consisting of the following (D), (E), and (F):

(D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17;

(E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several bases are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and

(F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.

[9] A microorganism transformed with a DNA encoding a pipecolic acid 4-hydroxylase protein selected from the group consisting of the following (D), (E), and (F):

(D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17;

(E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several bases are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and

(F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.

[10] The microorganism according to [9], which is selected from the group consisting of the genera Escherichia, Bacillus, Pseudomonas, and Corynebacterium.

Effect of the Invention

According to the present invention, various 4-hydroxy amino acids can be efficiently produced. In particular, optically active 4-hydroxy amino acids such as (4S)-hydroxy-L-pipecolic acid and (4S)-hydroxy-D-pipecolic acid, which are useful as intermediates of pharmaceuticals, can be efficiently produced at high optical purities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting L-proline with E. coli transformed with pFoPA4H.

FIG. 2 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting L-leucine with E. coli transformed with pFoPA4H.

FIG. 3 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting L-pipecolic acid with E. coli transformed with pFoPA4H.

FIG. 4 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting D-proline with E. coli transformed with pEnPA4H.

FIG. 5 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting (S)-2-aminobutyric acid with E. coli transformed with pEnPA4H.

FIG. 6 is a diagram showing the result of ¹H-NMR analysis of a reaction product obtained by reacting L-pipecolic acid with E. coli transformed with pFoPA4H (halftone image).

FIG. 7 is a diagram showing the result of ¹³C-NMR analysis of a reaction product obtained by reacting L-pipecolic acid with E. coli transformed with pFoPA4H (halftone image).

FIG. 8 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting (5S)-hydroxy-L-pipecolic acid with a cell-free extract obtained from E. coli transformed with pEnPA4H.

FIG. 9 is a diagram showing the result of LC-MS analysis of a reaction liquid obtained by reacting (4S)-hydroxy-L-proline with a cell-free extract obtained from E. coli transformed with pEnPA4H.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention is described below in detail.

<Pipecolic Acid 4-Hydroxylase, and Method for Producing 4-Hydroxy Amino Acid Using THE SAME>

The pipecolic acid 4-hydroxylase of the present invention comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, or a homologue sequence thereof having pipecolic acid 4-hydroxylase activity.

The amino acid sequences of SEQ ID NOs:2, 4, 6, 8, 10, and 12 are derived from the Fusarium oxysporum strain, Aspergillus oryzae RIB40 strain, Penicillium chrysogenum Wisconsin 54-1255 strain, Gibberella zeae (another name, Fusarium graminearum) PH-1 strain, Colletotrichum gloeosporioides Nara gc5 strain, and Aspergillus nidulans (another name, Emericella nidulans) FGSC A4 strain, respectively. These are the amino acid sequences of proteins identified as pipecolic acid 4-hydroxylase in the present invention. The amino acid sequences of SEQ ID NOs:16 and 18 are sequences obtained by analysis of DNAs directly collected from samples of soils in Japan, and are the amino acid sequences of proteins identified as pipecolic acid 4-hydroxylase in the present invention. Although the amino acid sequence of SEQ ID NO:16 shows a homology of 97% to a protein which is derived from Variovorax paradoxus EPS and whose function is unknown (GenBank accession No. YP_004156498), there is no report describing its function. Further, although the amino acid sequence of SEQ ID NO:18 shows a homology of 50% to a protein which is derived from Burkholderia sp A1 and whose function is unknown (GenBank accession No. WP_029951026), there is no report describing its function.

None of these sequences can be easily assumed to be a hydroxylase for pipecolic acid based only on its sequence information. The present invention identified them as pipecolic acid 4-hydroxylases for the first time.

A plurality of types of pipecolic acid 4-hydroxylases may be used.

In the present description, the pipecolic acid 4-hydroxylase activity means an activity which adds a hydroxyl group to the 4-position carbon atom of L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions, to produce trans-4-hydroxy-L-pipecolic acid. Such an activity can be confirmed by allowing, as an enzyme, the protein of interest, a cell(s) expressing the protein, or a treated product thereof to act in a reaction system containing L-pipecolic acid as a substrate and 2-oxoglutaric acid and iron(11) ions as cofactors, and then measuring the production of trans-4-hydroxy-L-pipecolic acid as described in the later-mentioned Examples.

Examples of the homologue of the pipecolic acid 4-hydroxylase comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 in the present invention include those comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, as long as the homologues have pipecolic acid 4-hydroxylase activity. Here, the term “one or several amino acids” means, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, especially preferably 1 to 5 amino acids.

The homologue may be a protein having a sequence that is not less than 80%, preferably not less than 90%, more preferably not less than 95%, identical to the full-length amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, or 18 as long as the homologue maintains pipecolic acid 4-hydroxylase activity.

The pipecolic acid 4-hydroxylase that may be used in the present invention can be obtained by purification from the microorganisms described above, or can be obtained by cloning a DNA encoding the pipecolic acid 4-hydroxylase by a known method such as PCR or hybridization, and allowing expression of the pipecolic acid 4-hydroxylase in an appropriate host.

Examples of the DNA encoding pipecolic acid 4-hydroxylase include DNAs comprising the nucleotide sequence represented by SEQ ID NOs:1, 3, 5, 7, 9, 11, 15, or 17.

The DNA encoding pipecolic acid 4-hydroxylase may also be a homologue of a DNA comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 as long as the DNA encodes a protein having pipecolic acid 4-hydroxylase activity. Examples of such a homologue include those comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several nucleotides are substituted, deleted, and/or added.

Here, the term “one or several nucleotides” means, for example, 1 to 300, preferably 1 to 150, more preferably 1 to 60, still more preferably 1 to 30, especially preferably 1 to 15 nucleotides.

The DNA homologue may be a DNA which hybridizes with the complementary strand of the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, as long as the DNA homologue encodes a protein having pipecolic acid 4-hydroxylase activity. Examples of the “stringent conditions” herein include conditions in which washing is carried out in the presence of 0.1×SSC (saline-sodium citrate) and 0.1% SDS (sodium dodecyl sulfate) at 60° C.

Those skilled in the art can obtain such a DNA homologue by appropriately introducing a substitution, deletion, insertion, and/or addition mutation(s) into the DNA of SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 by site-directed mutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982); Methods in Enzymol. 100, pp. 448 (1983); Molecular Cloning, PCR A Practical Approach IRL Press pp. 200 (1991)) or the like.

The amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 or a part thereof, or the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 or a part thereof may be subjected to homology search against a database provided by, for example, DNA Databank of JAPAN (DDBJ), to obtain amino acid information for the pipecolic acid 4-hydroxylase activity, or nucleotide sequence information of a DNA encoding it.

In the method for producing 4-hydroxy amino acid of the present invention, pipecolic acid 4-hydroxylase may be directly used in the reaction. However, it is preferred to use cells containing pipecolic acid 4-hydroxylase, a treated product of the cells, or a culture liquid obtained by culturing the cells.

The cells containing pipecolic acid 4-hydroxylase may be cells of a microorganism intrinsically having pipecolic acid 4-hydroxylase. However, it is preferred to use cells transformed with a gene encoding pipecolic acid 4-hydroxylase.

Examples of the treated product of the cells containing pipecolic acid 4-hydroxylase include treated cell products such as those obtained by treatment with an organic solvent such as acetone, dimethylsulfoxide (DMSO), or toluene, or with a surfactant, those obtained by freeze-drying, and those obtained by physical or enzymatic destruction; enzyme fractions of the cells obtained as a crude product or a purified product; and products prepared by immobilizing these on a carrier such as polyacrylamide gel and carrageenan gel.

By inserting the thus isolated DNA encoding pipecolic acid 4-hydroxylase into a known expression vector in a manner which allows expression of the pipecolic acid 4-hydroxylase, a pipecolic acid 4-hydroxylase-expressing vector is provided. By transforming host cells with this expression vector, a transformant to which a DNA encoding pipecolic acid 4-hydroxylase is introduced can be obtained. The transformant can also be obtained by incorporating a DNA encoding pipecolic acid 4-hydroxylase into the chromosomal DNA of a host by a method such as homologous recombination in a manner which allows expression of the pipecolic acid 4-hydroxylase.

Specific examples of the method for preparing the transformant include a method in which a DNA encoding pipecolic acid 4-hydroxylase is introduced into a plasmid vector, phage vector, or virus vector which can be stably present in a host cell such as a microorganism cell, and the constructed expression vector is introduced into the host cell, or the DNA is directly into the host genome, followed by allowing transcription and translation of the genetic information. In this process, an appropriate promoter is preferably linked 5′-upstream of the DNA, and, in addition, a terminator is preferably linked 3′-downstream of the DNA. Such a promoter and a terminator are not limited as long as they are a promoter and a terminator known to function in the cell used as a host. For example, “Fundamental Microbiology 8: Genetic Engineering, KYORITSU SHUPPAN CO., LTD.” describes details of vectors, promoters, and terminators that can be used in host microorganisms.

The host microorganism to be transformed for expression of pipecolic acid 4-hydroxylase is not limited as long as the host itself does not adversely affect the reaction of α-amino acid, and specific examples of the host microorganism include the following microorganisms:

Bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus, and the like whose host vector systems have been established.

Actinomycetes belonging to the genera Rhodococcus, Streptomyces, and the like whose host vector systems have been established.

Yeasts belonging to the genera Saccharomyces, Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Hansenula, Pichia, Candida, and the like whose host vector systems have been established.

Fungi belonging to the genera Neurospora, Aspergillus, Cephalosporium, Trichoderma, and the like whose host vector systems have been established.

The procedure for construction of the transformant, the method for construction of a recombinant vector suitable for the host, and the method for culturing the host can be carried out according to techniques commonly used in the fields of molecular biology, bioengineering, and genetic engineering (for example, methods described in Molecular Cloning).

The following are specific examples of preferred host microorganisms, and preferred methods of transformation, vectors, promoters, terminators, and the like for the microorganisms. The present invention is not limited to these examples.

For the genus Escherichia, especially Escherichia coli, examples of the plasmid vector include pBR and pUC plasmids, and examples of the promoter include promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc (fusion of lac and trp), and λ phage PL and PR. Examples of the terminator include terminators derived from trpA, phages, and rrnB ribosomal RNA.

For the genus Bacillus, examples of the vector include pUB110 plasmids and pC194 plasmids. Integration into the chromosome is also possible. Examples of the promoter and the terminator include those of genes of enzymes such as alkaline protease, neutral protease, and α-amylase.

For the genus Pseudomonas, examples of the vector include common host vector systems established in Pseudomonas putida, Pseudomonas cepacia, and the like; and a wide-host-range vector (containing genes required for autonomous replication derived from RSF1010 and the like) pKT240, which is based on a plasmid involved in degradation of toluene compounds, TOL plasmid (Gene, 26, 273-82 (1983)).

For the genus Brevibacterium, especially Brevibacterium lactofermentum, examples of the vector include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). Examples of the promoter and the terminator include promoters and terminators used in E. coli.

For the genus Corynebacterium, especially Corynebacterium glutamicum, examples of the vector include plasmid vectors such as pCS11 (JP 57-183799 A) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)).

For the genus Saccharomyces, especially Saccharomyces cerevisiae, examples of the vector include YRp, YEp, YCp, and YIp plasmids. Examples of promoters and terminators which may be used include those of the genes of enzymes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase.

For the genus Schizosaccharomyces, examples of the vector include the plasmid vector derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Shuzo Co., Ltd., and can be easily used.

In terms of the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, and the like are the best-studied species among fungi. Plasmids, and integration into the chromosome are applicable to these species, and promoters derived from extracellular protease and amylase can be used (Trends in Biotechnology 7, 283-287 (1989)).

Among the microorganisms described above, examples of especially preferred microorganisms include microorganisms belonging to the genera Escherichia, Bacillus, Pseudomonas, and Corynebacterium having no glycosylation ability.

Host-vector systems other than the above-described systems have also been established for various microorganisms, and those systems may be used as appropriate.

Various host-vector systems have been established for plants and animals besides microorganisms. In particular, systems for allowing expression of a large amount of foreign protein in an animal such as an insect including silkworm (Nature 315, 592-594 (1985)), or in a plant such as rapeseed, maize, or potato; and systems using cell-free protein synthesis systems such as E. coli cell-free extracts and wheat germs; have been established, and may be preferably used.

By reacting pipecolic acid 4-hydroxylase, cells containing the enzyme, a treated product of the cells, or a culture liquid of the cells, with the reaction substrate α-amino acid in the presence of 2-oxoglutaric acid and iron(II) ions, 4-hydroxy amino acid is produced.

Here, the α-amino acid as the reaction substrate is not limited as long as it has a hydrogen atom which can be substituted with a hydroxyl group at 4-position. Examples of the α-amino acid include compounds represented by the following General Formula (I). Examples of the 4-hydroxy amino acid include compounds represented by the following General Formula (II). Each of the α-amino acid and the 4-hydroxy amino acid is preferably an L-isomer or a D-isomer, but may also be a racemic mixture.

In General Formulae (I) and (II), each of R¹, R², and R⁵ represents a hydrogen atom or C₁-C₃ alkyl, and each of R³ and R⁴ represents a hydrogen atom, C₁-C₃ alkyl, or hydroxyl. Alternatively, R² may bind to R⁵ or the nitrogen atom of the amino group to form a ring structure.

Specific examples of the type of the α-amino acid include valine, leucine, isoleucine, proline (in which R² is bound to the nitrogen atom of the amino group to form a five-membered ring), α-aminobutyric acid, norvaline, norleucine, pipecolic acid (in which R² is bound to R⁵ to form a six-membered ring), 3-hydroxyproline, 3-hydroxypipecolic acid, and 5-hydroxypipecolic acid. The α-amino acid is especially preferably pipecolic acid.

The reaction is carried out in an aqueous medium containing: the α-amino acid; 2-oxoglutaric acid; iron(II) ions; and the pipecolic acid 4-hydroxylase or the cells containing it, the treated product of the cells, or the culture product of the cells; or carried out in a mixture of the aqueous medium and an organic solvent.

Examples of the aqueous medium include water and buffers. Here, the buffers are not limited as long as the activity of the pipecolic acid 4-hydroxylase is not inhibited thereby. Examples of the buffers include phosphate buffer and MES (2-Morpholinoethanesulfonic acid) buffer. Examples of the organic solvent include those in which the reaction substrate is highly soluble, such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, and dimethyl sulfoxide. Other examples of the organic solvent include ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane, which are effective for removal of reaction by-products, and the like.

The reaction substrate, α-amino acid, is usually used at a substrate concentration within the range of 0.01% w/v to 90% w/v, preferably 0.1% w/v to 30% w/v. The reaction substrate may be added at once when the reaction is started, but is preferably added continuously or intermittently from the viewpoint of reducing the influence of substrate inhibition of the enzyme, if any, and increasing the concentration of the product accumulated.

The number of moles of the 2-oxoglutaric acid required for the reaction is normally equivalent to, or higher than, that of the substrate, preferably equivalent to, or up to 1.2-fold higher than, that of the substrate. The 2-oxoglutaric acid may be added at once when the reaction is started, but is preferably added continuously or intermittently from the viewpoint of reducing the inhibitory action on the enzyme, if any, and increasing the concentration of the product accumulated. Alternatively, an inexpensive compound that can be metabolized by the host, such as glucose or L-glutamic acid, may be added instead of 2-oxoglutaric acid to allow metabolism of the compound by the host, and 2-oxoglutaric acid produced during this process may be used for the reaction.

The iron(II) ions required for the reaction are used within the range of usually 0.01 mmol/L to 100 mmol/L, preferably 0.1 mmol/L to 10 mmol/L. The iron(II) ions are usually added at once when the reaction is started. Further addition of iron(II) ions is also effective in cases where the iron(II) ions decrease during the reaction due to oxidation into iron(III) ions or formation of a precipitate. In cases where a sufficient amount of iron(II) ions are already contained in the pipecolic acid 4-hydroxylase, the cells containing the enzyme, the treated product of the cells, or the culture liquid of the cells, the addition of iron(II) ions is not required.

The reaction is carried out at a reaction temperature of usually 4° C. to 60° C., preferably 10° C. to 45° C., at a pH of usually 3 to 11, preferably 5 to 8. The reaction time is usually about 1 hour to about 72 hours.

The amount of the cells and/or the treated cell product to be added to the reaction liquid is as follows. In cases of addition of the cells, the cells are added to the reaction liquid such that the cell concentration is usually about 0.1% w/v to about 50% w/v, preferably 1% w/v to 20% w/v, in terms of the wet cell weight. In cases of using the treated cell product, the specific activity of the enzyme in the treated cell product is determined, and the treated cell product is added such that the cell concentration described above is achieved.

After completion of the reaction, the 4-hydroxy amino acid produced by the method of the present invention may be subjected to separation of microbial cells, proteins, and/or the like in the reaction liquid by centrifugation, membrane treatment, and/or the like, and then to purification by an appropriate combination of, for example, extraction with an organic solvent such as 1-butanol or tert-butanol, distillation, column chromatography using an ion-exchange resin, silica gel, or the like, crystallization at the isoelectric point, and/or crystallization with monohydrochloride, dihydrochloride, calcium salt, or the like.

EXAMPLES

The present invention is described below in more detail by way of Examples, but the present invention is not limited to the Examples.

Example 1 Construction of Plasmid Expressing Pipecolic Acid 4-Hydroxylase Gene (1) Gene Cloning

Based on a gene sequence encoding a hypothetical protein FOXB_08233 derived from Fusarium oxysporum Fo5176, primers BOF1 and BOR1 for amplification of full-length homologous genes for the hypothetical protein FOXB_08233 were designed and synthesized. The nucleotide sequences of the primers are shown in SEQ ID NOs:13 and 14.

Fusarium oxysporum c8D was cultured overnight in a potato dextrose liquid medium (manufactured by Becton, Dickinson and Company, Japan). From the microbial cells obtained, chromosomal DNA was prepared using a DNeasy Blood & Tissue Kit (manufactured by QIAGEN).

Using the thus prepared chromosomal DNA derived from each microbial strain as a template, and the oligonucleotides of SEQ ID NOs:13 and 14 as primers, polymerase chain reaction (PCR) was carried out to amplify a DNA fragment of about 1 kbp. The PCR was carried out using Tks Gflex DNA polymerase (manufactured by Takara Bio Inc.) according to the conditions described in the manufacturer's instruction. The temperature program was as follows: the temperature was kept at 95° C. for 1 minute, and then 35 cycles of (98° C., 10 seconds; 56.5° C., 15 seconds; and 68° C., 40 seconds) were carried out, followed by keeping the temperature at 72° C. for 3 minutes to complete the reaction. The result of analysis of the DNA sequence obtained is shown in SEQ ID NO:1, and the amino acid sequence encoded by this DNA sequence is shown in SEQ ID NO:2.

(2) Preparation of Expression Plasmid

The DNA fragment obtained in (1) was digested with restriction enzymes BamHI and HindIII, and introduced into a plasmid vector pQE80L (manufactured by QIAGEN) digested with the restriction enzymes BamHI and HindIII, using a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.). The plasmid obtained is hereinafter referred to as pFoPA4H. In addition, the DNA sequences of SEQ ID NOs:3, 5, 7, 9, and 11 were synthesized by DNA2.0, Inc., and inserted into an expression plasmid pJexpress411 by the same manufacturer. The plasmids obtained are hereinafter referred to as pAoPA4H, pPcPA4H, GzPA4H, pCgPA4H, and pEnPA4H.

Example 2 Evaluation of Pipecolic Acid 4-Hydroxylase Activity

The six kinds of plasmids obtained in Example 1, pFoPA4H, pAoPA4H, pPcPA4H, GzPA4H, pCgPA4H, and pEnPA4H, were used for transformation of E. coli (Escherichia coli) Rosetta 2 (DE3) (manufactured by Merck Millipore Corporation) according to an ordinary method. Each recombinant E. coli obtained was cultured with shaking at 30° C. using liquid LB medium supplemented with 50 mg/L kanamycin, 15 mg/L chloramphenicol, and 1 mmol/L IPTG (isopropyl β-D-1-thiogalactopyranoside) for 20 hours, and the cultured bacterial cells were collected.

The bacterial cells obtained were subjected to reaction at 20° C. at pH 6 for 21 hours in 50 mmol/L MES (2-morpholinoethanesulfonic acid) buffer supplemented with 15 mmol/L 2-oxoglutaric acid, 5 mmol/L ascorbic acid, 5 mmol/L tris(2-carboxyethyl)phosphine hydrochloride, 0.5 mmol/L FeSO₄.7H₂O, 3 mmol/L citric acid, and 10 mmol/L each substrate compound.

The reaction liquids after the reaction were analyzed by high-performance liquid chromatography (HPLC) under the following conditions.

Column: CHIRALPAK AD-3 (4.6×250 mm, manufactured by Daicel Corporation) Eluent:hexane:ethanol:trifluoroacetic acid=95:5:0.1 Flow rate: 0.8 ml/min.

Temperature: 30° C. Detection: UV 210 nm Example 3 Identification of Reaction Products

Each reaction liquid obtained in Example 2 was derivatized using the Waters AccQ.Tag method, and subjected to separation analysis using a high-performance liquid chromatograph-mass spectrometer (LC-MS) under the following conditions to measure amino acid reaction products.

Column: XBridge C18 5 μm (2.1×150 mm, manufactured by Waters Corporation) Eluent A: ammonium acetate (10 mmol/L, pH 5) Eluent B: methanol (0 to 0.5 min. (0%→1%), 0.5 to 18 min. (1%→5%), 18 to 19 min. (5%→9%), 19 to 29.5 min. (9%→17%), 29.5 to 40 min. (17%→60%), 40 to 43 min. (60%)) Flow rate: 0.3 ml/min.

Temperature: 30° C.

Detection: mass spectrometer

For the reaction liquids obtained using the six kinds of E. coli, substrate amino acids for which appearance of a new peak was found in the LC-MS analysis are shown in Table 1. Since the molecular weights of these new reaction products were found to be higher by 16 compared to the molecular weights of their substrates, it is thought that all of the new reaction products are compounds produced by addition of an oxygen atom to their substrates.

TABLE 1 Substrate amino acid (the amount of increase in the molecular weight Plasmid of the reaction product) pFoPA4H L-pipecolic acid (16), L-proline (16), L-leucine (16), D-norleucine (16) pAoPA4H L-pipecolic acid (16), L-proline (16), L-leucine (16), D-norleucine (16) pPcPA4H L-pipecolic acid (16), L-leucine (16) pGzPA4H L-pipecolic acid (16), L-leucine (16) pCgPA4H L-pipecolic acid (16), L-leucine (16) pEnPA4H L-pipecolic acid (16), D-pipecolic acid (16), L-proline (16), D-proline (16), L-leucine (16), L-valine (16), D-valine (16), L-norleucine (16), D-norleucine (16), (S)-2-Aminobutyric acid, L-norvaline (16) (1) The result of LC-MS analysis of the reaction liquid containing E. coli transformed with pFoPA4H and L-proline is shown in FIG. 1. Since the peak of the reaction product showed the same retention time as that of a standard substance for trans-4-hydroxy-L-proline, the reaction product was assumed to be trans-4-hydroxy-L-proline. The same result was obtained in the cases where E. coli transformed with pAoPA4H or pEnPA4H was used. (2) The result of LC-MS analysis of the reaction liquid containing E. coli transformed with pFoPA4H and L-leucine are shown in FIG. 2. Since the peak of the reaction product showed the same retention time as that of a standard substance for 4-hydroxy-L-leucine, the reaction product was assumed to be trans-4-hydroxy-L-leucine. The same result was obtained in the cases where E. coli transformed with pAoPA4H, pPcPA4H, GzPA4H, pCgPA4H, or pEnPA4H was used. (3) A reaction product obtained by reacting L-pipecolic acid with E. coli transformed with pFoPA4H was collected by high-performance liquid chromatography (HPLC) under the following conditions. Column: TSKgel Amide80 (7.8×300 mm, manufactured by Tosoh Corporation) Eluent: ammonium acetate (10 mmol/L, pH 5):acetonitrile=15:85 Flow rate: 2.3 ml/min.

Temperature: 40° C. Detection: UV 210 nm

The eluate containing the reaction product was collected, and dried under reduced pressure using a centrifugal evaporator. The resulting residue was suspended in heavy water, and subjected to measurement of the magnetic resonance spectrum.

Based on the chemical shift values obtained as a result of ¹H-NMR (FIG. 6) and ¹³C-NMR (FIG. 7), the correlation obtained as a result of HH-COSY and CH-HMQC analyses, and information from a literature (Molnar, T. et al., 2008, Bioorg. Med. Chem. Lett., 18, 6290), the reaction product was identified as trans-4-hydroxy-L-pipecolic acid.

The result of LC-MS analysis of the reaction liquid containing E. coli transformed with pFoPA4H and L-pipecolic acid is shown in FIG. 3. Elution of the peak of the reaction product, trans-4-hydroxy-L-pipecolic acid, occurred at 10.9 minutes. The same result was obtained in the cases where E. coli transformed with pAoPA4H, pPcPA4H, GzPA4H, pCgPA4H, or pEnPA4H was used.

(4) The result of LC-MS analysis of the reaction liquid containing E. coli transformed with pEnPA4H and D-proline is shown in FIG. 4. Since the peak of the reaction product showed the same retention time as that of a standard substance for cis-4-hydroxy-L-proline, the reaction product was assumed to be cis-4-hydroxy-D-proline. (5) The result of LC-MS analysis of the reaction liquid containing E. coli transformed with pEnPA4H and (S)-2-aminobutyric acid is shown in FIG. 5. Since the peak of the reaction product showed the same retention time as that of a standard substance for L-homoserine, the reaction product was assumed to be L-homoserine.

Example 4 Evaluation of Activity of Pipecolic Acid 4-Hydroxylase for Hydroxylation to Hydroxy Amino Acid

Using cell-free extracts obtained from the cells obtained in Example 2, reaction was carried out at 20° C. at pH 6 for 21 hours in 50 mmol/L MES buffer supplemented with 15 mmol/L 2-oxoglutaric acid, 5 mmol/L ascorbic acid, 5 mmol/L tris(2-carboxyethyl)phosphine hydrochloride, 0.5 mmol/L FeSO₄.7H₂O, 3 mmol/L citric acid, and 10 mmol/L each substrate compound. The reaction liquids after the reaction were analyzed by high-performance liquid chromatography (HPLC) under the conditions described in Example 2.

For reaction liquids using two kinds of cell-free extracts, substrate hydroxy amino acids for which appearance of a new peak was found in the LC-MS analysis are shown in Table 2. Since the molecular weights of these new reaction products were found to be higher by 16 compared to the molecular weights of their substrates, it is thought that all of the new reaction products are dihydroxy amino acids produced by addition of an oxygen atom to hydroxy amino acids.

TABLE 2 Substrate amino acid (the amount of increase in the molecular weight Plasmid of the reaction product) pFoPA4H (5S)-hydroxy-L-pipecolic acid (16) pAoPA4H (5S)-hydroxy-L-pipecolic acid (16) pPcPA4H (5S)-hydroxy-L-pipecolic acid (16) pGzPA4H (5S)-hydroxy-L-pipecolic acid (16) pCgPA4H (5S)-hydroxy-L-pipecolic acid (16) pEnPA4H (5S)-hydroxy-L-pipecolic acid (16), (3R)-hydroxy-L-proline (16)

Example 5 Construction of Plasmids Expressing Pipecolic Acid 4-Hydroxylase Gene

From results of sequence analysis of DNA directly collected from soil samples, SEQ ID NOs:15 and 17, which could be genes for amino acid hydroxylases, were discovered. The amino acid sequences encoded by the DNA sequences are shown in SEQ ID NOs:16 and 18.

From each DNA sequence, a DNA fragment of about 1 kbp was amplified by PCR. Each fragment obtained was introduced into pQE80L (manufactured by QIAGEN) according to the method described in Example 1. The plasmids obtained are hereinafter referred to as pVsPA4H and pBsPA4H, respectively.

Example 6 Evaluation of Pipecolic Acid 4-Hydroxylase Activity

Using the two kinds of plasmids pVsPA4H and pBsPA4H obtained in Example 5, transformation of E. coli (Escherichia coli) Rosetta 2 (DE3) (manufactured by Merck Millipore Corporation) was carried out according to an ordinary method. Each recombinant E. coli obtained was cultured with shaking at 30° C. using liquid LB medium supplemented with 50 mg/L ampicillin, 15 mg/L chloramphenicol, and 1 mmol/L IPTG for 20 hours, and the cultured bacterial cells were collected. Using cell-free extracts obtained from the collected cells, reaction was carried out at 20° C. at pH 6 for 21 hours in 50 mmol/L MES buffer supplemented with 15 mmol/L 2-oxoglutaric acid, 5 mmol/L ascorbic acid, 5 mmol/L tris(2-carboxyethyl)phosphine hydrochloride, 0.5 mmol/L FeSO₄.7H₂O, 3 mmol/L citric acid, and 10 mmol/L substrate compound (L-pipecolic acid).

The reaction liquids after the reaction were analyzed by the above-mentioned high-performance liquid chromatography (HPLC). As a result, a compound having the same molecular weight as trans-4-hydroxy-L-pipecolic acid was found in both reaction liquids. 

1. A pipecolic acid 4-hydroxylase protein having an activity to react with L-pipecolic acid in the presence of 2-oxoglutaric acid and iron(II) ions, to produce trans-4-hydroxy-L-pipecolic acid.
 2. The pipecolic acid 4-hydroxylase protein according to claim 1, selected from the group consisting of the following (A), (B), and (C): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18; (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18 except that one or several amino acids are deleted, substituted, and/or added, and having pipecolic acid 4-hydroxylase activity; and (C) a polypeptide comprising an amino acid sequence that is not less than 80% identical to the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8, 10, 12, 16, or 18, and having pipecolic acid 4-hydroxylase activity.
 3. The pipecolic acid 4-hydroxylase protein according to claim 2, which is produced as a recombinant protein by a host having no glycosylation ability.
 4. A method for producing 4-hydroxy amino acid, said method comprising reacting said pipecolic acid 4-hydroxylase protein according to claim 1, a cell(s) comprising said protein, a treated product of said cell(s), and/or a culture liquid obtained by culturing said cell(s), with α-amino acid to produce 4-hydroxy amino acid.
 5. The method for producing 4-hydroxy amino acid according to claim 4, wherein said α-amino acid is represented by the following General Formula (I):

(wherein each of R¹, R², and R⁵ represents a hydrogen atom or C₁-C₃ alkyl; each of R³ and R⁴ represents a hydrogen atom, C₁-C₃ alkyl, or hydroxyl; and alternatively R² may bind to R⁵ or the nitrogen atom of the amino group to form a ring structure), and said 4-hydroxy amino acid is represented by the following General Formula (II):

(wherein each of R¹, R², and R⁵ represents a hydrogen atom or C₁-C₃ alkyl; each of R³ and R⁴ represents a hydrogen atom, C₁-C₃ alkyl, or hydroxyl; and alternatively R² may bind to R⁵ or the nitrogen atom of the amino group to form a ring structure).
 6. The method for producing 4-hydroxy amino acid according to claim 4, wherein said α-amino acid is pipecolic acid, and said 4-hydroxy amino acid is 4-hydroxy-L-pipecolic acid.
 7. The method for producing 4-hydroxy amino acid according to claim 4, wherein said cell comprising pipecolic acid 4-hydroxylase is a cell transformed with a DNA encoding pipecolic acid 4-hydroxylase protein.
 8. The method for producing 4-hydroxy amino acid according to claim 7, wherein said DNA encoding pipecolic acid 4-hydroxylase protein is selected from the group consisting of the following (D), (E), and (F): (D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17; (E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and (F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.
 9. A microorganism transformed with a DNA encoding a pipecolic acid 4-hydroxylase protein selected from the group consisting of the following (D), (E), and (F): (D) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17; (E) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 except that one or several nucleotides are substituted, deleted, and/or added, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity; and (F) a DNA comprising a nucleotide sequence which hybridizes with the complementary strand of the nucleotide sequence represented by SEQ ID NO:1, 3, 5, 7, 9, 11, 15, or 17 under stringent conditions, and encoding a polypeptide having pipecolic acid 4-hydroxylase activity.
 10. The microorganism according to claim 9, which is selected from the group consisting of the genera Escherichia, Bacillus, Pseudomonas, and Corynebacterium. 